Pregnane X receptor activation induces liver enlargement and regeneration and simultaneously promotes the metabolic activity of CYP3A1/2 and CYP2C6/11 in rats.

Human pregnane X receptor (PXR) is critical for regulating the expression of key drug-metabolizing enzymes such as CYP3A and CYP2C. Our recent study revealed that treatment with rodent-specific PXR agonist pregnenolone-16α-carbonitrile (PCN) significantly induced hepatomegaly and promoted liver regeneration after two-thirds partial hepatectomy (PHx) in mice. However, it remains unclear whether PXR activation induces hepatomegaly and liver regeneration and simultaneously promotes metabolic function of the liver. Here, we investigated the metabolism activity of CYP1A2, CYP3A1/2 and CYP2C6/11 during PXR activation-induced liver enlargement and regeneration in rats after cocktail dosing of CYP probe drugs. For PCN-induced hepatomegaly, a notable increase in the metabolic activity of CYP3A1/2 and CYP2C6/11, as evidenced by the plasma exposure of probe substrates and the AUC ratios of the characteristic metabolites to its corresponding probe substrates. The metabolic activity of CYP1A2, CYP3A1/2 and CYP2C6/11 decreased significantly after PHx. However, PCN treatment obviously enhanced the metabolic activity of CYP2C6/11 and CYP3A1/2 in PHx rats. Furthermore, the protein expression levels of CYP3A1/2 and CYP2C6/11 in liver were up-regulated. Taken together, this study demonstrates that PXR activation not only induces hepatomegaly and liver regeneration in rats, but also promotes the protein expression and metabolic activity of the PXR downstream metabolizing enzymes such as CYP3A1/2 and CYP2C6/11 in the body.

Fructose Metabolism in Tumor Endothelial Cells Promotes Angiogenesis by Activating AMPK Signaling and Mitochondrial Respiration.

Angiogenesis is vital for tumor growth and metastasis. Emerging evidence suggests that metabolic reprogramming in endothelial cells (EC) may affect angiogenesis. Here, we showed that multiple regulators in the fructose metabolism pathway, especially fructose transporter SLC2A5 and fructose-metabolizing enzyme ketohexokinase (KHK), were upregulated in tumor endothelial cells from hepatocellular carcinoma (HCC). In mouse models with hepatoma xenografts or with Myc/sgp53-induced liver cancer, dietary fructose enhanced tumor angiogenesis, tumor growth, and metastasis, which could be attenuated by treatment with an inhibitor of SLC2A5. Furthermore, vessel growth was substantially increased in fructose-containing Matrigel compared with PBS-Matrigel. Inhibiting fructose metabolism in EC cells in vivo using EC-targeted nanoparticles loaded with siRNA against KHK significantly abolished fructose-induced tumor angiogenesis. Fructose treatment promoted the proliferation, migration, and tube formation of ECs and stimulated mitochondrial respiration and ATP production. Elevated fructose metabolism activated AMPK to fuel mitochondrial respiration, resulting in enhanced EC migration. Fructose metabolism was increased under hypoxic conditions as a result of HIF1α-mediated upregulation of multiple genes in the fructose metabolism pathway. These findings highlight the significance of fructose metabolism in ECs for promoting tumor angiogenesis. Restricting fructose intake or targeting fructose metabolism is a potential strategy to reduce angiogenesis and suppress tumor growth. SIGNIFICANCE: Fructose metabolism in endothelial cells fuels mitochondrial respiration to stimulate tumor angiogenesis, revealing fructose metabolism as a therapeutic target and fructose restriction as a dietary intervention for treating cancer.

Mitochondrial Micropeptide STMP1 Enhances Mitochondrial Fission to Promote Tumor Metastasis.

Micropeptides are a recently discovered class of molecules that play vital roles in various cellular processes, including differentiation, proliferation, and apoptosis. Here, we sought to identify cancer-associated micropeptides and to uncover their mechanistic functions. A micropeptide named short transmembrane protein 1 (STMP1) that localizes at the inner mitochondrial membrane was identified to be upregulated in various cancer types and associated with metastasis and recurrence of hepatocellular carcinoma. Both gain- and loss-of-function studies revealed that STMP1 increased dynamin-related protein 1 (DRP1) activation to promote mitochondrial fission and enhanced migration of tumor cells. STMP1 silencing inhibited in vivo tumor metastasis in xenograft mouse models. Overexpression of STMP1 led to redistribution of mitochondria to the leading edge of cells and enhanced lamellipodia formation. Treatment with a DRP1 inhibitor abrogated the promotive effect of STMP1 on mitochondrial fission, lamellipodia formation, and tumor cell migration in vitro and metastasis in vivo. Furthermore, STMP1 interacted with myosin heavy chain 9 (MYH9), the subunit of nonmuscle myosin II, and silencing MYH9 abrogated STMP1-induced DRP1 activation, mitochondrial fission, and cell migration. Collectively, this study identifies STMP1 as a critical regulator of metastasis and a novel unit of the mitochondrial fission protein machinery, providing a potential therapeutic target for treating metastases. SIGNIFICANCE: This study identifies the mitochondrial micropeptide STMP1 as a regulator of metastasis that promotes mitochondrial fission and tumor cell migration via DRP1 and MYH9.

Downregulation of a mitochondrial micropeptide, MPM, promotes hepatoma metastasis by enhancing mitochondrial complex I activity.

We and others have shown that MPM (micropeptide in mitochondria) regulates myogenic differentiation and muscle development. However, the roles of MPM in cancer development remain unknown. Here we revealed that MPM was downregulated significantly in human hepatocellular carcinoma (HCC) tissues and its decrease was associated with increased metastasis potential and HCC recurrence. Gain- and loss-of-function investigations disclosed that in vitro migration/invasion and in vivo liver/lung metastasis of hepatoma cells were repressed by restoring MPM expression and increased by silencing MPM. Mechanism investigations revealed that MPM interacted with NDUFA7. Mitochondrial complex I activity was inhibited by overexpressing MPM and enhanced by siMPM, and this effect of siMPM was attenuated by knocking down NDUFA7. The NAD+/NADH ratio, which was regulated by complex I, was reduced by MPM but increased by siMPM. Treatment with the NAD+ precursor nicotinamide abrogated the inhibitory effect of MPM on hepatoma cell migration. Further investigations showed that miR-17-5p bound to MPM and inhibited MPM expression. miR-17-5p upregulation was associated with MPM downregulation in HCC tissues. These findings indicate that a decrease in MPM expression may promote hepatoma metastasis by increasing mitochondrial complex I activity and the NAD+/NADH ratio.

Identification of a TGF-ß/SMAD/lnc-UTGF positive feedback loop and its role in hepatoma metastasis.

Aberrant activation of the TGF-ß/SMAD signaling pathway is often observed in hepatocellular carcinoma (HCC). Whether lncRNA regulates the TGF-ß/SMAD signaling remains largely unknown. Here, we identified an oncogenic lncRNA that was upregulated in HCC and was transcriptionally induced by TGF-ß (named lnc-UTGF, lncRNA upregulated by TGF-ß). Upon TGF-ß stimulation, SMAD2/3 bound to the lnc-UTGF promoter and activated lnc-UTGF expression. In turn, the TGF-ß/SMAD signaling was augmented by overexpressing lnc-UTGF, but was inhibited by silencing lnc-UTGF. Mechanism investigations revealed that lnc-UTGF interacted with the mRNAs of SMAD2 and SMAD4 via complementary base-pairing, resulting in enhanced stability of SMAD2/4 mRNAs. These data suggest a novel TGF-ß/SMAD/lnc-UTGF positive feedback circuitry. Subsequent gain- and loss-of-function analyses disclosed that lnc-UTGF promoted the migration and invasion of hepatoma cells, and this effect of lnc-UTGF was attenuated by repressing SMAD2/4 expression or by mutating the SMAD2/4-binding sites in lnc-UTGF. Studies using mouse models further confirmed that in vivo metastasis of hepatoma xenografts was inhibited by silencing lnc-UTGF, but was enhanced by ectopic expression of lnc-UTGF. The lnc-UTGF level was positively correlated with the SMAD2/4 levels in xenografts. Consistently, we detected an association of lnc-UTGF upregulation with increase of SMAD2, SMAD4, and their metastasis effector SNAIL1 in human HCC. And high lnc-UTGF level was also significantly associated with enhanced metastasis potential, advanced TNM stages, and worse recurrence-free survival. Conclusion: there exists a lnc-UTGF-mediated positive feedback loop of the TGF-ß signaling and its deregulation promotes hepatoma metastasis. These findings may provide a new therapeutic target for HCC metastasis.

Dual and opposing roles of the androgen receptor in VETC-dependent and invasion-dependent metastasis of hepatocellular carcinoma.

BACKGROUND & AIMS: Contradictory roles of the androgen receptor (AR) in hepatocellular carcinoma (HCC) metastasis have been reported. We have shown that VETC (vessels encapsulating tumor clusters) mediates invasion-independent metastasis, whereas VETC- HCCs metastasize in an invasion-dependent manner. Herein, we aimed to reveal the roles of AR in HCC metastasis. METHODS: Mouse xenograft models, clinical samples, and cell models were used. RESULTS: AR expression was significantly lower in HCCs with a VETC pattern, portal vein tumor thrombus, endothelium-coated microemboli or high recurrence rates. Overexpressing AR in VETC+ hepatoma cells suppressed VETC formation and intrahepatic metastasis but promoted pulmonary metastasis of mouse xenografts. AR decreased the transcription of Angiopoietin-2 (Angpt2), a factor essential for VETC formation, by binding to the Angpt2 promoter. The roles of AR in inhibiting VETC formation and intrahepatic metastasis were attenuated by restoring Angpt2 expression, suggesting that AR may repress VETC-dependent intrahepatic metastasis by inhibiting Angpt2 expression and VETC formation. On the other hand, AR upregulated Rac1 expression, promoted lamellipodia formation and increased cell migration/invasion. A Rac1 inhibitor abrogated the AR-mediated promotion of migration/invasion and pulmonary metastasis of VETC+ hepatoma cells, but did not affect the AR-mediated inhibition of intrahepatic metastasis. Furthermore, an AR inhibitor decreased Rac1 expression and attenuated both intrahepatic and pulmonary metastasis of VETC- xenografts, an effect which was abrogated by restoring Rac1 expression. These data indicate that AR may facilitate the lung metastasis of VETC+ HCCs and both the liver/lung metastases of VETC- HCCs by upregulating Rac1 expression and then promoting migration/invasion. CONCLUSION: AR plays dual and opposing roles in VETC-dependent and invasion-dependent metastasis, which highlights the complex functions of AR and the importance of individualized cancer therapy. LAY SUMMARY: In this study, we uncovered the dual and opposing roles of the androgen receptor in VETC (vessels encapsulating tumor clusters)-dependent and invasion-dependent metastasis of hepatocellular carcinoma (HCC). We elucidated the underlying mechanisms of these processes, which provided novel insights into the complex regulatory network of the androgen receptor in HCC metastasis and may have important implications for precision medicine.

Functional long non-coding RNAs in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a prevalent human malignancy with high morbidity worldwide. Hepatocarcinogenesis is a complex multistep process, and its underlying molecular mechanisms remain largely unknown. Recently, long non-coding RNAs (lncRNAs), a class of newly discovered molecules, have been revealed as essential regulators in the development of HCC. HCC-associated lncRNAs affect multiple malignant phenotypes by modulating gene expression or protein activity. Moreover, the dysregulation of lncRNAs in the liver is also associated with diseases predisposing to HCC, such as chronic viral infection, nonalcoholic steatohepatitis, and liver fibrosis/cirrhosis. A deeper understanding of the lncRNA regulatory network in the multistep processes of HCC development will provide new insights into the diagnosis and treatment of HCC. In this review, we introduce the biogenesis and function of lncRNAs and summarize recent knowledge on how lncRNAs regulate the malignant hallmarks of HCC, such as uncontrolled cell proliferation, resistance to cell death, metabolic reprogramming, immune escape, angiogenesis, and metastasis. We also review emerging insights into the role of lncRNAs in HCC-associated liver diseases. Finally, we discuss the potential applications of lncRNAs as early diagnostic biomarkers and therapeutic targets.

A hMTR4-PDIA3P1-miR-125/124-TRAF6 Regulatory Axis and Its Function in NF kappa B Signaling and Chemoresistance.

BACKGROUND AND AIMS: DNA damage-induced NF-κB activation is a major obstacle to effective antitumour chemotherapy. Long noncoding RNAs (lncRNAs) that regulate chemoresistance of cancer cells remain largely unknown. This study aimed to characterize the lncRNAs that may affect chemotherapy sensitivity. APPROACH AND RESULTS: We found that lncRNA PDIA3P1 (protein disulfide isomerase family A member 3 pseudogene 1) was up-regulated in multiple cancer types and following treatment with DNA-damaging chemotherapeutic agents, like doxorubicin (Dox). Higher PDIA3P1 level was associated with poorer recurrence-free survival of human hepatocellular carcinoma (HCC). Both gain-of-function and loss-of-function studies revealed that PDIA3P1 protected cancer cells from Dox-induced apoptosis and allowed tumor xenografts to grow faster and to be more resistant to Dox treatment. Mechanistically, miR-125a/b and miR-124 suppressed the expression of tumor necrosis factor receptor-associated factor 6 (TRAF6), but PDIA3P1 bound to miR-125a/b/miR-124 and relieved their repression on TRAF6, leading to activation of the nuclear factor kappa B (NF-κB) pathway. Consistently, the effect of PDIA3P1 inhibition in promoting Dox-triggered apoptosis was antagonized by silencing the inhibitor of κBα (IκBα) or overexpressing TRAF6. Administration of BAY 11-7085, an NF-κB inhibitor attenuated PDIA3P1-induced resistance to Dox treatment in mouse xenografts. Moreover, up-regulation of PDIA3P1 was significantly correlated with elevation of TRAF6, phosphorylated p65, or NF-κB downstream anti-apoptosis genes in human HCC tissues. These data indicate that enhanced PDIA3P1 expression may confer chemoresistance by acting as a microRNA sponge to increase TRAF6 expression and augment NF-κB signaling. Subsequent investigations into the mechanisms of PDIA3P1 up-regulation revealed that human homologue of mRNA transport mutant 4 (hMTR4), which promotes RNA degradation, could bind to PDIA3P1, and this interaction was disrupted by Dox treatment. Overexpression of hMTR4 attenuated Dox-induced elevation of PDIA3P1, whereas silencing hMTR4 increased PDIA3P1 level, suggesting that Dox may up-regulate PDIA3P1 by abrogating the hMTR4-mediated PDIA3P1 degradation. CONCLUSION: There exists a hMTR4-PDIA3P1-miR-125/124-TRAF6 regulatory axis that regulates NF-κB signaling and chemoresistance, which may be exploited for anticancer therapy.

Vessels That Encapsulate Tumor Clusters (VETC) Pattern Is a Predictor of Sorafenib Benefit in Patients with Hepatocellular Carcinoma.

Sorafenib is the most recommended first-line systemic therapy for advanced hepatocellular carcinoma (HCC). Yet there is no clinically applied biomarker for predicting sorafenib response. We have demonstrated that a vascular pattern, named VETC (Vessels that Encapsulate Tumor Clusters), facilitates the release of whole tumor clusters into the bloodstream; VETC-mediated metastasis relies on vascular pattern, but not on migration and invasion of cancer cells. In this study, we aimed to explore whether vascular pattern could predict sorafenib benefit. Two cohorts of patients were recruited from four academic hospitals. The survival benefit of sorafenib treatment for patients with or without the VETC pattern (VETC+ /VETC- ) was investigated. Kaplan-Meier analyses revealed that sorafenib treatment significantly reduced death risk and prolonged overall survival (OS; in cohort 1/2, P = 0.004/0.005; hazard ratio [HR] = 0.567/0.408) and postrecurrence survival (PRS; in cohort 1/2, P = 0.001/0.002; HR = 0.506/0.384) in VETC+ patients. However, sorafenib therapy was not beneficial for VETC- patients (OS in cohort 1/2, P = 0.204/0.549; HR = 0.761/1.221; PRS in cohort 1/2, P = 0.121/0.644; HR = 0.728/1.161). Univariate and multivariate analyses confirmed that sorafenib treatment significantly improved OS/PRS in VETC+ , but not VETC- , patients. Further mechanistic investigations showed that VETC+ and VETC- HCCs displayed similar levels of light chain 3 (LC3) and phosphorylated extracellular signal-regulated kinase (ERK) in tumor tissues (pERK) or endothelial cells (EC-pERK), and greater sorafenib benefit was consistently observed in VETC+ HCC patients than VETC- irrespective of levels of pERK/EC-pERK/LC3, suggesting that the different sorafenib benefit between VETC+ and VETC- HCCs may not result from activation of Raf/mitogen-activated protein kinase kinase (MEK)/ERK and vascular endothelial growth factor (VEGF)A/VEGF receptor 2 (VEGFR2)/ERK signaling or induction of autophagy. Conclusion: Sorafenib is effective in prolonging the survival of VETC+ , but not VETC- , patients. VETC pattern may act as a predictor of sorafenib benefit for HCC.

MicroRNA-125b Promotes Hepatic Stellate Cell Activation and Liver Fibrosis by Activating RhoA Signaling.

miR-125b is frequently dysregulated in different diseases. Activation of hepatic stellate cells (HSCs) is a critical event during liver fibrogenesis. However, the function and its underlying mechanism of miR-125b in HSC activation and liver fibrosis are still unknown. Here, we showed that miR-125b was upregulated in HSCs, but not in hepatocytes, during hepatic fibrogenesis in vivo and upon culture activation in vitro. Inhibition of miR-125b suppressed the expression of profibrogenic genes in culture-activated primary HSCs and reduced the basal and transforming growth factor ß (TGF-ß)-induced alpha-smooth muscle actin (α-SMA) expression and cell contraction of the immortalized HSC cell line. In contrast, ectopic expression of miR-125b promoted α-SMA expression and HSC contraction. Moreover, antagonizing miR-125b in vivo significantly alleviated liver fibrosis in CCl4-treated mice. Mechanistically, overexpression of miR-125b in HSCs enhanced RhoA activity by directly targeting StAR-related lipid transfer (START) domain containing 13 (Stard13), a RhoA-specific GTPase-activating protein, whereas knockdown of miR-125b abrogated RhoA activation. Furthermore, inhibition of RhoA or its downstream molecules, Mrtf-A and Srf, attenuated the miR-125b-induced α-SMA expression and HSC contraction. Therefore, our findings identify a miR-125b-Stard13-RhoA-α-SMA signaling cascade in HSCs and highlight its importance in hepatic fibrosis.

Hepatocellular Carcinoma Cell-Secreted Exosomal MicroRNA-210 Promotes Angiogenesis In Vitro and In Vivo.

We previously found that 19 microRNAs (miRNAs) significantly increased in the sera of hepatocellular carcinoma (HCC) patients. Here, we evaluated whether these miRNAs were secreted by HCC cells and contributed to tumor angiogenesis. High level of miR-210-3p (miR-210) was detected in the exosomes isolated from the sera of HCC patients and the conditioned media of hepatoma cells. Higher miR-210 level in serum was correlated with higher microvessel density in HCC tissues. Moreover, the HCC cell-secreted exosomes promoted in vitro tubulogenesis of endothelial cells, which was strengthened by overexpressing miR-210 in HCC cells but was attenuated by repressing miR-210 or DROSHA in HCC cells. This pro-tubulogenesis effect by HCC exosomes was also abrogated by antagonizing miR-210 in endothelial cells. Subsequent in vivo studies revealed that Matrigel plug and subcutaneous tumor xenografts treated with HCC cell-derived exosomal miR-210 displayed much more vessels. Furthermore, exosomal miR-210 could be delivered into endothelial cells and directly inhibited the expression of SMAD4 and STAT6, resulting in enhanced angiogenesis. Collectively, HCC cell-secreted exosomal miR-210 may be transferred into endothelial cells and thereby promotes tumor angiogenesis by targeting SMAD4 and STAT6. Our findings identify a novel mechanism of HCC angiogenesis and highlight the biological importance of exosomal miR-210.

Hepatoma cell-secreted exosomal microRNA-103 increases vascular permeability and promotes metastasis by targeting junction proteins.

Increased vascular permeability facilitates metastasis. Emerging evidence indicates that secreted microRNAs (miRNAs) may mediate the crosstalk between cancer and stromal cells. To date, whether and how secreted miRNAs affect vascular permeability remains unclear. Based on deep sequencing and quantitative PCR, we found that higher level of serum miR-103 was associated with higher metastasis potential of hepatocellular carcinoma (HCC). The in vitro endothelial permeability and transendothelial invasion assays revealed that the conditioned media or exosomes derived from high miR-103-expressing hepatoma cells increased the permeability of endothelial monolayers, but this effect was attenuated if exosome secretion of hepatoma cells was blocked by silencing ALIX and HRS or if miR-103 within hepatoma or endothelial cells was antagonized. Most importantly, pretreating endothelial monolayers with exosomes that were from stable miR-103-expressing hepatoma cells facilitated the transendothelial invasion of tumor cells, and this role of exosomes was abrogated by inhibiting miR-103 in endothelial cells. Further in vivo analyses disclosed that mice with xenografts of stable miR-103-expressing hepatoma cells exhibited higher vascular permeability in tumor, higher level of exosomal miR-103 and greater number of tumor cells in blood circulation, and increased rates of hepatic and pulmonary metastases, compared to control mice. Mechanism investigations revealed that hepatoma cell-secreted miR-103 could be delivered into endothelial cells via exosomes, and then attenuated the endothelial junction integrity by directly inhibiting the expression of VE-Cadherin (VE-Cad), p120-catenin (p120) and zonula occludens 1. Moreover, miR-103 could also promote tumor cell migration by repressing p120 expression in hepatoma cells. CONCLUSION: Hepatoma cell-secreted exosomal miR-103 increases vascular permeability and promotes tumor metastasis by targeting multiple endothelial junction proteins, which highlights secreted miR-103 as a potential therapeutic target and a predictive marker for HCC metastasis. (Hepatology 2018).

Effect of smoking on high-grade cervical cancer in women on the basis of human papillomavirus infection studies.

PURPOSE: We aimed, in the present study, to measure the risk related to the high-grade cervical intraepithelial neoplasia grade 3 (CIN3) or worse (CIN3+) or worse/high-grade squamous intraepithelial lesions with respect to changes in human papillomavirus (HPV) and smoking status. MATERIALS AND METHODS: A structured interview underwent for 7129 women. Then, we obtained their cervical cells and subjected to HPV testing. High-risk HPV infected and "no prevalent" cervical disease infected women were followed for cervical lesions up to 12 years (at baseline; n = 1531). Hazard ratios (HRs) for diagnosis of CIN3 (or worse) or worse/high-grade intraepithelial lesions were calculated along with the corresponding 95% confidence intervals (CIs). RESULTS: Among high-risk HPV-positive women, the conditions of long-term (more than 8 years) smokers and heavy (18 or more cigarettes/day) smokers are highly responsible for the increased risk for CIN3 or CIN3+. In the cases of persistent HPV-infected women, heavy smoking led to a higher risk for CIN3+ than those women who never smoked (HR, 2.31; 95% CI, 1.12-4.16). CONCLUSION: We concluded here that smoking leads to an enhanced risk of high-grade cervical lesions in persistent high-risk HPV-infected women. This makes a good understanding of smoking's role in cervical cancer.

Regulatory Role of the MicroRNA-29b-IL-6 Signaling in the Formation of Vascular Mimicry.

Vascular mimicry (VM) is a critical complement for microcirculation and is implicated in tumor progression. We showed that IL-6 derived from tumor cells and stroma cells promoted tumor cells to form a VM structure, whereas blocking the IL-6 signaling by RNA interference, IL-6-neutralizing antibody, or STAT3 inhibitor suppressed the VM formation of tumor cells. Mechanism investigations revealed that IL-6 stimulated VM formation by activating STAT3, in turn upregulating VE-cadherin expression and MMP2 activity. Further analyses identified a positive association between the activation of IL-6-STAT3 signaling and the formation of the VM structure in human HCC tissues. However, miR-29b repressed the expression of STAT3 and MMP2 by directly binding to the 3' UTRs of their mRNAs. Consistently, both gain- and loss-of-function analyses showed that miR-29b suppressed tumor cells to form tube structures in vitro. The in vivo studies further disclosed that intratumoral injection of the miR-29b-expressing viruses significantly inhibited the IL-6-promoted VM formation in mouse xenografts, and downregulation of miR-29b was correlated with the presence of VM in human HCC tissues. This study elucidates a miR-29b-IL-6 signaling cascade and its role in VM formation, which provide potential targets for cancer therapy.

Vascular mimicry formation is promoted by paracrine TGF-ß and SDF1 of cancer-associated fibroblasts and inhibited by miR-101 in hepatocellular carcinoma.

Vascular mimicry (VM) describes the phenomenon that tumor cells but not endothelial cells form vascular-like channels, which provide blood perfusion for tumor tissues. VM is associated with tumor growth, metastasis and worse survival of different cancers. The mechanisms of VM formation remain largely unknown. We showed that the conditioned medium of cancer-associated fibroblast (CM-CAF) promoted tumor cells to form capillary-like structure in vitro. Consistently, co-implantation of CAFs with tumor cells significantly enhanced VM formation in mouse xenografts, and higher amount of CAFs was found in VM+ human HCC tissues compared to VM- ones. However, the CM-CAF-promoted VM formation was attenuated when TGF-ß or SDF1 signaling was abrogated. Similar to CM-CAF, recombinant TGF-ß1 and SDF1 induced VM formation. We further disclosed that the CAF-secreted TGF-ß and SDF1 enhanced the expression of VE-cadherin, MMP2 and laminin5γ2 via TGF-ßR1 and CXCR4 in tumor cells, thereby promoted VM formation. Moreover, tumor cells with high activity of self-sustaining TGF-ß signaling displayed strong capability of VM formation. Subsequent investigations showed that miR-101, which was down-regulated in both tumor cells and CAFs, suppressed the CAF-promoted VM formation in vitro and in vivo. Gain- and loss-of-function analyses revealed that miR-101 attenuated TGF-ß signaling transduction by targeting TGF-ßR1 and Smad2 in tumor cells, and simultaneously abrogated SDF1 signaling by suppressing SDF1 expression in CAFs and inhibiting VE-cadherin expression in tumor cells. Our findings suggest that the miR-101-TGF-ß/SDF1-VE-cadherin/MMP2/LAMC2 networks regulate VM formation and represent the potential targets for cancer therapy.

MicroRNAs miR-125b and miR-100 suppress metastasis of hepatocellular carcinoma by disrupting the formation of vessels that encapsulate tumour clusters.

We have previously shown that vessels that encapsulated tumour cluster (VETC), a prevalent vascular pattern in hepatocellular carcinoma (HCC), facilitates the entry of the whole tumour cluster into the bloodstream in an invasion-independent manner, and that angiopoietin 2 (Angpt2), the levels of which are increased in HCC cells, is essential for VETC formation. However, the mechanisms underlying VETC formation remains unclear. Herein, we characterized miR-125b and miR-100 as novel VETC suppressors by using human HCC specimens, and cell and animal models. We showed that reduced expression of either miR-125b or miR-100 in human HCC tissues was significantly associated with the presence of VETC, venous invasion of tumour cells, and the occurrence of endothelium-coated microemboli. To confirm the role of miR-125b and miR-100 in VETC formation and HCC metastasis, cell lines with stable miR-125b and miR-100 expression were established by using human VETC-2 cells and mouse Hepa1-6 cells, the hepatoma cells that developed xenografts with VETC patterns. Our results showed that expression of miR-125b or miR-100 in VETC-2 and Hepa1-6 cells dramatically reduced VETC formation in xenografts, and consequently inhibited in vivo metastasis, suggesting that miR-125b and miR-100 may attenuate metastasis by repressing VETC formation. Further investigation revealed that miR-125b directly suppressed the expression of Angpt2 by binding to its 3'-untranslated region, whereas miR-100 reduced the protein level of Angpt2 by targeting mechanistic target of rapamycin (MTOR) and blocking the MTOR-p70S6K signalling pathway. Moreover, the suppressive effect of miR-125b and miR-100 on VETC formation was abrogated by injecting Angpt2-expressing viruses into xenografts. Taken together, our findings imply that miR-125b and miR-100 negatively regulate Angpt2 expression through different mechanisms, in turn inhibit VETC formation, and consequently abrogate the VETC-dependent metastasis of hepatoma cells. This study uncovers new regulatory mechanisms of VETC formation, identifies novel functions of miR-125b and miR-100, and provides new targets for antimetastasis therapy of HCC. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Fibrotic microenvironment promotes the metastatic seeding of tumor cells via activating the fibronectin 1/secreted phosphoprotein 1-integrin signaling.

The seeding of tumor cells is a critical step in the process of metastasis, but whether and how the microenvironment of target organs affects metastatic seeding remain largely unknown. Based on cell and mouse models, we found that the metastatic seeding and outgrowth of tumor cells were significantly enhanced in fibrotic lungs. The conditioned medium from both fibrotic lungs and the fibrotic lung-derived fibroblasts (CM-FLF) had a strong activity to chemoattract tumor cells and to inhibit the apoptosis of tumor cells. Subsequent investigations revealed that the levels of fibronectin 1 (FN1) and secreted phosphoprotein 1 (SPP1) were significantly increased in fibrotic lungs. Silencing of FN1 in the fibrotic lung-derived fibroblasts dramatically decreased the chemoattracting activity of CM-FLF, while silencing of FN1 or SPP1 in fibroblasts attenuated the anti-apoptosis activity of CM-FLF. Moreover, the CM-FLF-induced apoptosis resistance or chemotaxis of tumor cells was attenuated when ITGAV, the common receptor of FN1 and SPP1, was silenced by RNA interference or blocked by GRGDS treatment in tumor cells. Consistently, ITGAV silencing or GRGDS treatment significantly inhibited the seeding and outgrowth of tumor cells in fibrotic lungs in vivo. Collectively, we suggest that fibrotic microenvironment may enhance the metastatic seeding of tumor cells in the lung by chemoattracting tumor cells and inhibiting their apoptosis via activating the FN1/SPP1-ITGAV signaling. These findings give a novel insight into the regulatory mechanisms of cancer metastasis and provide a potential target for anti-metastasis therapy.